Phytochemical profile and antiproliferative effect of Ficus crocata extracts on triple-negative breast cancer cells

Background Some species of the Ficus genus show pharmacological activity, including antiproliferative activity, in cell lines of several cancer Types. ficus crocata is distributed in Mexico and used in traditional medicine, as it is believed to possess anti-inflammatory, analgesic, and antioxidant properties. However, as of yet, there are no scientific reports on its biological activity. This study aims to evaluate the phytochemical profile of F. crocata leaf extracts and their effects on breast cancer MDA-MB-231 cells proliferation. Moreover, the study aims to unearth possible mechanisms involved in the decrease of cell proliferation. Methods The extracts were obtained by the maceration of leaves with the solvents hexane, dichloromethane, and acetone. The phytochemical profile of the extracts was determined using gas chromatography coupled with mass analysis. Cell proliferation, apoptosis, and cell cycle analysis in MDA-MB-231 cells were determined using a Crystal violet assay, MTT assay, and Annexin-V/PI assay using flow cytometry. The data were analyzed using ANOVA and Dunnett’s test. Results The hexane (Hex-EFc), dichloromethane (Dic-EFc), and acetone (Ace-EFc) extracts of F. crocata decreased the proliferation of MDA-MB-231 cells, with Dic-EFc having the strongest effect. Dic-EFc was fractioned and its antiproliferative activity was potentiated, which enhanced its ability to induce apoptosis in MDA-MB-231 cells, as well as increased p53, procaspase-8, and procaspase-3 expression. Conclusions This study provides information on the biological activity of F. crocata extracts and suggests their potential use against triple-negative breast cancer.


Background
Breast cancer is the most common cancer and the main cause of death for women worldwide. It represents about 12% of all new cancer cases and 25% of all cancers in women [1]. Triple-negative breast cancer (TNBC) represents 10-20% of all breast carcinomas [2] and is characterized by no expression of estrogen receptors (ER) and progesterone receptors (PR), and the lack of overexpression of the HER2 protein. Thus, the tumoral cells do not respond to hormone therapy, and it is considered the subtype with the worst prognosis among breast cancer cases [3][4][5]. Among the different drugs used in cancer, many of these are derived from plants and some are utilized in their natural form or with structural modifications, such as polyphenols (e.g., resveratrol [6], epicathechin [7], and epigallocatechin gallate [8]) and flavonoids (e.g., quercetin [9], silibinin [10], and oncamex [11]), which, in cancer cells, have demonstrated the ability to induce apoptosis [12][13][14][15].

Preparation of extracts and fractionation
Leaves of F. crocata (100 g) were dried and ground, and then successively macerated (sequential extraction) with hexane, dichloromethane, and acetone solvents (reactive-grade, 500 mL during 24 h, three times). The macerated material was filtered, and the organic phase was evaporated in a rotary evaporator (Digital Rotary Evaporator Model 410) at 60°C and 80 rpm. Hexane (1.51% yield; Hex-EFc), and dichloromethane (0.94% yield; Dic-EFc), and acetone (11.75% yield; Ace-EFc) extracts were stored at − 20°C and protected from light.

Gas chromatography coupled with mass analysis (GC-MS)
GC-MS analyses were carried in triplicate out on an Agilent 6890 series gas chromatograph equipped with a mass selective detector 5973 N (USA). The experimental conditions of GC-MS system were as follows [64]: HP-5MS capillary nonpolar column (30 m, ID: 0.20 mm, film thickness: 0.25 μm). The carrier gas was helium at flow rate of 1.0 mL/min. In the gas chromatography part, the temperature program (oven temperature) was 50°C raised to 230°C at 2°C/min and the injection volume was 1 μL. Samples were dissolved in dichloromethane. All results were compared by using NIST/EPA/NIH Mass Spectral library version 1.7a/ChemStation.

Cell culture and exposure to extracts
MDA-MB-231 cells (ATCC® HTB-26) were cultured in Dulbecco's Modified Eagle Medium Formula 12 (DMEM/F12) supplemented with 5% Fetal Bovine Serum (FBS), 1% antibiotic (ampicillin/streptomycin), and incubated at 37°C in a 5% CO 2 atmosphere and at 95% humidity. Cells were synchronized with basal medium without FBS for 24 h and exposed to 0-80 μg mL − 1 of F. crocata extracts for 24-48 h. DiMethyl SulfOxide (DMSO) was used as the diluent of extracts (Vehicle). As a positive control, cells were treated with 100 μM Cytarabine (Ara-C), which is known to induce cell death [65]. As a negative control, cells were treated with 5% FBS to induce cell proliferation. All tests were performed in triplicate at three independent times.

Cell growth in monolayer (crystal violet assay)
A total of 15 × 10 3 cells were plated on 24-well plates (Corning) and cultured in DMEM/F12 medium supplemented with 5% FBS for 24 h. Cells were treated with 0, 5, 10, 20, 40, and 80 μg mL − 1 of F. crocata extracts for 24 and 48 h, fixed after treatment in 4% formaldehyde for 5 min, and the cell morphology was observed using an inverted microscope (EVOS Cell Imaging System; Thermo Scientific). Later, the cells were stained with 0.5% Crystal violet, the excess dye was washed with water and PBS (Phosphate-Buffered Saline) and the stain was extracted with 10% acetic acid. The relative cell density in the monolayer was determined by measuring the optical density (OD) of each well at 600 nm in a biophotometer (Eppendorf Model RS-2312 DH 8.5 mm).

MTT assay
The percentage of viable cells was evaluated using the MTT cell proliferation colorimetric assay (CT02, Millipore Corp., Bedford, MA, USA) according to the manufacturer's instructions. Briefly, in a 96-well plate, 1 × 10 4 MDA-MB-231 cells per well were cultured for 24 h with DMEM/F12 medium with 5% FBS and subsequently with basal medium for 24 h to synchronize the cells in the G1 phase and promote a homogeneous cellular response. Then, the treatment with 0-80 μg mL − 1 of F. crocata extracts or fractions was applied for 24 and 48 h. After the treatment, the medium containing the extracts was replaced by fresh basal medium and 100 μL of the MTT reagent was added for 4 h. The formazan crystals were diluted with isopropanol, and the OD of the supernatant was obtained at a wavelength of 540 nm using a biophotometer (Eppendorf Model RS-2312 DH 8.5 mm). The half-maximal inhibitory concentration (IC 50 ) was calculated through the linear eq. (Y = mX + b) using GraphPad prism software v6.0.

Analysis of apoptosis
In order to determine the cell population in apoptosis after the treatment with F. crocata extracts, the Fluorescein Isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I (556,547; Beckton Dickinson) was used according to the manufacturer's instructions. Briefly, MDA-MB-231 cells were seeded at a density of 2 × 10 5 cells/well in a six-well plate for 24 h with DMEM and 5% FBS. The cells were synchronized with basal medium for 24 h and subsequently exposed to Dic-EFc (0, 20, 40, and 80 μg mL − 1 ) or A9, A12, and A13-Dic-EFc fractions (80 μg mL − 1 ) for 48 h. Cells were collected by trypsinization followed by washing with PBS, and staining with FITC Annexin V and propidium iodide (PI) for 15 min in the dark. They were then immediately analyzed by flow cytometry (FACSCanto II; Beckton Dickinson, USA). The FITC Annexin V-and PI-negative cells were considered viable cells. Cells in early apoptosis were FITC Annexin V-positive and PI-negative, while cells in late apoptosis were both FITC Annexin V-and PIpositive.

Analysis of cell cycle arrest
MDA-MB-231 cells were seeded at a density of 2 × 10 5 cells/well in a six-well plate and incubated for 24 h in an incubator at 37°C with a 5% CO 2 atmosphere. The cells were synchronized in basal medium for 24 h and subsequently exposed to Dic-EFc (0, 20, 40, and 80 μg mL − 1 ) or A9, A12, and A13-Dic-EFc fractions (80 μg mL − 1 ) for 48 h. Cells were collected by trypsinization followed by washing with PBS, centrifugation, and fixing with ethanol. Subsequently, the cells were centrifuged and resuspended in cold PBS. A total of 20 μg mL − 1 RNAsa was added for 30 min and, the cells were stained with Propidium Iodide (PI) in the dark at room temperature for 15 min. The immunofluorescence of PI was analyzed by flow cytometry (FACSCanto II; Beckton Dickinson).

Statistical analysis
Data analysis was performed using the GraphPad Prism version 7.0 statistical software. The data were shown as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used with Dunnett's test. A statistically significant difference was considered when p < 0.05.
Leaf extracts of F. crocata decreased the proliferation of MDA-MB-231 cells To evaluate the antiproliferative activity of F. crocata extracts on MDA-MB-231 breast cancer cells, crystal violet and MTT assays were performed. It was observed that Hex-EFc, Dic-EFc, and Ace-EFc decreased the number of cells in the monolayer in a concentration-and timedependent manner. At 24 h of treatment, only 40 and 80 μg mL − 1 concentrations decreased the cell density compared to the untreated control cells (p < 0.001). The effect of extracts was greater at 48 h, showing a lower cell density in the monolayer at all of the tested concentrations and, decreasing the number of cells as the concentration increased (p < 0.001) (Fig. 1). In addition, Dic-EFc and Ace-EFc induced morphologic changes in MDA-MB-231 cells, such as a decrease in cell size, a rounded shape, and the formation of intracellular vacuoles suggestive of apoptosis (Fig. 2). These cellular changes were evident at 48 h with 5-, 10-, and 20-μg mL − 1 concentrations, mainly with Dic-EFc. Figure 2 shows the effect of the extracts at 20 μg mL − 1 after 48 h of treatment.
In order to validate the data observed with the Crystal violet assay, the effect of the extracts on cell proliferation was determined by MTT assay in order to consider only metabolically viable cells. After 24 h of treatment, a significant decrease in cell proliferation was observed with ≥5 μg mL − 1 of Dic-EFc, ≥10 μg mL − 1 of Ace-EFc, and ≥ 20 μg mL − 1 of Hex-EFc compared to untreated control cells. After prolonging exposure to extracts for 48 h, Hex-EFc, Dic-EFc, and Ace-EFc decreased cell proliferation at all tested concentrations compared to control cells (p < 0.001). However, this effect was more evident with Dic-EFc, which decreased the percentage of viable cells by more than 50% with 10-80 μg mL − 1 (p < 0.001) (Fig. 3). Antiproliferative activity at 48 h was greater for Dic-EFc (IC 50 : 32.43 μg mL − 1 ), followed by Ace-EFc (IC 50 : 78.49 μg mL − 1 ), and Hex-EFc (IC 50 : 164.05 μg mL − 1 ) (Fig. S4). In addition, the effect of DIC-EFc on viability of MCF-10A non-tumor cells was preliminary evaluated, and only 320 μg mL − 1 of extract showed cytotoxic effect at 48 h of treatment (Fig. S5).
The dichloromethane extract of F. crocata induced apoptosis and cell cycle arrest in MDA-MB-231 cells To determine possible mechanisms to reduce the proliferation of MDA-MB-231 cells exposed to the dichloromethane extract of F. crocata, an apoptosis assay and cell cycle analysis were performed. Only the concentrations of Dic-EFc (20-80 μg mL − 1 ) and its fractions (80 μg mL − 1 ) that reduced around 50% or more of cell proliferation were analyzed. It was observed that the Dic-EFc treatment induced apoptosis in 4.3 and 7.7% of the cell population with 20 μg mL − 1 and 40 μg mL − 1 , respectively, while 80 μg mL − 1 increased the apoptotic population to 19.3% (4.3 and 15% in early and late apoptosis, respectively). Interestingly, the fractionation of Dic-EFc components enhanced the ability of the extract to induce apoptosis in MDA-MB-231 cells. It was observed that A9-Dic-EFc, A12-Dic-EFc, and A13-Dic-EFc at 80 μg mL − 1 all induced apoptosis. The A9-Dic-EFc fraction showed the highest percentage of apoptotic cells with 53% (30.7% in early apoptosis and 22.3% in late apoptosis), followed by the A12-Dic-EFc and A13-Dic-EFc fractions, with 51.9 and 48.8%, respectively (Fig. 5). Moreover, a decrease was observed in the cell population in the S and G2/M phases of the cell cycle, and an increase during the sub-G0 phase was noted after treatment with Dic-EFc and its fractions A9, A12, and A13. A highlighted effect was observed with fraction A9, with more than 50% of the cell population occurring during the sub-G0 phase (Fig. 6). This high percentage in the sub-G0 phase was consistent with the apoptosis results observed with the Annexin/PI assay. Exposure to A12-Dic-EFc and A13-Dic-EFc fractions caused an accumulation of G1 phase cells of 62 and 53%, respectively, suggesting that metabolites in these fractions induce a G1-phase cell cycle arrest.
The dichloromethane extract of F. crocata increased the p53, procaspase-8 and procaspase-3 expression MDA-MB-231 cells were treated with 0-80 μg mL − 1 of Dic-EFc and 80 μg mL − 1 of the A9-Dic-EFc fraction to determine changes in expression of proteins such as p53, procaspase-8, and procaspases-3, which are associated with apoptosis and the cell cycle (Fig. 7). It was observed that the Dic-EFc and A9 fraction increased p53 expression at 24 h of treatment (Fig. 7b), although it markedly decreased p53 expression at 48 h (Fig. 7f). The increase in p53 expression could be associated with the diminution of cell number in the G1 and S phases of the cell cycle. With respect to caspase expression, in Western blot assays, we observed bands around 32 kDa and 55 kDa, which correspond to procaspase-3 and procaspase-8, respectively ( Fig. 7a and e). Dic-EFc increased procaspase-8 expression at 48 h of treatment only at the 80 μg mL − 1 concentration (Fig. 7g). Minor concentrations (20-40 μg mL − 1 ) of Dic-EFc increased procaspase-3 expression at 24 h and 48 h of treatment ( Fig. 7d and h). On the other hand, the A9-Dic-EFc fraction increased procaspase-8 expression at 24 h and 48 h of treatment ( Fig. 7c and g), and procaspase-3 expression at 48 h (Fig. 7h). It is possible that accumulated procaspase-3 and procaspase-8 are being processed into active forms-i.e., caspase 8 and caspase 3, respectivelywhich contribute to the progression of apoptosis induced mainly by treatment with the A9-Dic-EFc fraction in MDA-MB-231 cells.

Discussion
TNBC represents 15-20% of breast cancer cases and is related to poor prognosis, metastasis, and death. This type of breast tumor does not respond to conventional chemotherapy because the cells do not express ER and PR and lack overexpression of the HER2 protein, thus representing the sub-type with worst prognosis among breast cancer cases [66]. Current research is focused on the chemical compounds of plants and their effect on cellular models of cancer [14]. It has been observed that natural products obtained from plants exhibit antiproliferative, antimigratory, and anti-invasive have effects on cancer cells and could be used as adjuvant therapies in cancer [12][13][14][15]. Studies conducted in species of the Ficus genus have revealed the greatest antiproliferative potential in cervical, hepatic, leukemia, lung, and coloncancer cell lines [43,45,47,50]. It has been described that this activity is due to the presence of compounds such as alkaloids, flavonoids, coumarins, phenols, steroids, terpenoids, and triterpenoids [16, 18, 21, 23-26, 39, 40, 51, 52, 54, 56, 58, 59]. In Mexico, Ficus species are distributed throughout the country [60,61,67], yet there have been no studies, to our knowledge, on the chemical composition or biological properties of the Ficus species distributed in Mexico. In this study, we analyzed the phytochemical profile of leaf extracts of F. crocata and their effect on the cell proliferation of TNBC MDA-MB-231 cells. This is the first study, to our knowledge, to evaluate the cytotoxic activity of leaf extracts of F. crocata. We observed that Hex-EFc, Dic-EFc, and Ace-EFc decreased the proliferation of MDA-MB-231 cells, with Ace-EFc and mainly Dic-EFc being the most active. An interesting observation in this study was that Dic-EFc did not show a cytotoxic effect on MCF-10A non-tumor cells at the same concentrations that affected the viability of MDA-MB-231 cells (5-80 μg mL − 1 ), which supports the possible use of extracts of F. crocata as an alternative therapy against breast tumors. However, this observation must be analyzed further. These observations are in agreement with other studies, which reported a decrease of cell viability in the breast cancer cell lines T47D and MDA-MB-231 exposed to the leaf extracts of Ficus septica Burm. and Ficus carica [56,68].
To separate compounds into groups based on their polarity, Dic-EFc was fractionated and we observed that the A9-Dic-EFc, A12-Dic-EFc, and A13-Dic-EFc fractions exerted a greater effect in terms of decreasing the viability of MDA-MB-231 cells. A9-Dic-EFc was the most effective of the fractions, as compared to Dic-EFc. This effect suggests that isolation of compounds increased their biological activity. The phytochemical composition of Dic-EFc and the A9-Dic-EFc, A12-Dic-EFc, and A13-Dic-EFc fractions revealed the presence of alkaloids, coumarins, lignans, anthraquinones, phenols, terpenoids, and triterpenoids. The presence of these compounds has already been reported for the Ficus genus, and it has been demonstrated that these compounds possess biological activity [16,46,56,59]. We observed that the exposure of MDA-MB-231 cells to F. crocata extracts induced morphological changes such as a decrease in cell size, a rounded shape, and the formation of intracellular vacuoles. In a concentrationdependent manner, Dic-EFc and A9-Dic-EFc decreased the cell population in the S and G2/M phases of the cell cycle and increased the cell population in the sub-G0, which was consistent with the increase of apoptotic cells that could explain the morphologic changes in the cells and the decrease in cell viability. The apoptotic effect of Dic-EFc was potentiated with the A9-Dic-EFc, A12-Dic-EFc, and A13-Dic-EFc fractions, inducing apoptosis in around 50% of the cell population. In this regard, we observed that Dic-EFc and the A9-Dic-EFc fraction increased p53 expression at 24 h of treatment, which could be associated with apoptosis and cell cycle arrest, but curiously, p53 expression markedly decreased after of 48 h with 80 μg mL − 1 of Dic-EFc and A9-Dic-EFc. This increase and a subsequent decrease in the expression of (2020) 20:191 p53 has also been observed in another study with HCT116 cells treated with betulinic acid (BA), in which the treatment also induced apoptosis, suggesting that other proteins could be involved in BA-induced apoptosis in addition to p53 [69]. Moreover, in breast cancer cell lines MCF-7 and MDA-MB-231, it was observed that Vernonia amygdalina (VA) extracts induced apoptosis and cell cycle arrest in a p53-independent manner. Interestingly, these authors observed that VA increased p53 expression in a time-dependent manner in MCF-7 cells, whereas in MDA-MB-231, p53 expression decreased after 48 h [70]. We observed a similar event in our study. On the other hand, the A9-Dic-EFc fraction increased the expression of procaspase-8 and procaspase-3, which are initiator and executioner caspases, respectively, involved in the extrinsic apoptosis pathway [71]. These proteins could be processed in their active form and induce apoptosis, which would explain the increase in the apoptotic cell population induced mainly by the A9-Dic-EFc fraction in MDA-MB-231 cells. Dic-EFc also increased procaspase-8 expression at 48 h of treatment at an 80 μg mL − 1 concentration, while 20-40 μg mL − 1 concentrations increased procaspase-3 expression, which could be associated with the slight increase in the apoptotic cell population under these treatment conditions. It is probable that the apoptosis induced by the extracts of F. crocata and its fractions in MDA-MB-231 cells is through an p53-independent pathway, such as PUMA protein for example, which is known to be a powerful apoptosis inducer associated or not with p53 [72]. However, this hypothesis should still be tested. Our observations are consistent with those of other studies. Zhang et al. (2018) reported that F. carica extracts induce apoptosis and cell cycle arrest at the S phase in MDA-MB-231 cells, increasing the expression of genes that promote apoptosis and the regulation of the cell cycle, such as BAX, TP53, and TP21 [68]. Similarly, in a study by Choudhari et al., the authors reported that the aqueous extract of Ficus religiosa induces cell cycle arrest in the G1 phase in SiHa cells, accompanied by an increase in the expression of p53, p21, and pRb proteins. Moreover, in HeLa cells, the extract induces apoptosis through an increase in intracellular Ca 2+ , leading to the loss of mitochondrial membrane potential, the release of cytochrome-C, and an increase in caspase-3 expression [47]. It has also been reported that the flavonoid quercetin induces apoptosis in HeLa cells, promoting the accumulation of reactive oxygen species (ROS), upregulating proapoptotic proteins such as cytochrome C, Apaf-1, and caspases, and downregulating the antiapoptotic proteins Bcl-2 and survivin [73,74].
The primary compound in Dic-EFc was lupeol, a triterpene also present in Ace-EFc and Hex-EFc, although in smaller proportions (Table 1). Triterpenes were only present in the A9-Dic-EFc fraction, which suggests that lupeol was only present in A9-Dic-EFc and not in the A12-and A13-Dic-EFc fractions. It has been reported that lupeol induces apoptosis and cell cycle arrest in several cancer cell lines. For example, in pancreatic cancer cells, lupeol induces apoptosis by decreasing the levels of p-AKT and p-ERK, as well as cell cycle arrest in the G0/G1 phase, by upregulating P21 and P27 and downregulating cyclin D1 proteins [75]. A similar effect was observed in osteosarcoma cells MNNG/HOS and MG-63 [76]. In gallbladder carcinoma GBC-SD cells, lupeol was shown to induce apoptosis and inhibit invasion by downregulating the activity of p-EGFR and MMP-9 [77]. In non-small cell lung cancer cells, it was observed that lupeol inhibits the phosphorylation of EGFR by binding to its tyrosine kinase domain and reducing STAT3 phosphorylation, which contributes to the induction of apoptosis [78]. In the hepatocarcinoma cell lines SMMC7721 and HepG2 as well as lung carcinoma A427 cells, lupeol was shown to induce low expression of the antiapoptotic protein Bcl-2 and upregulate the proapoptotic protein BAX [79,80]. Considering the previously noted observations, it is possible that the metabolites present in F. crocata extracts, such as lupeol, probably in synergy with other metabolites, induce cell cycle arrest and apoptosis in MDA-MB-231 cells, altering the expression of cell cycle regulatory proteins such as p53, p21, p27, and cyclins, as well as the expression of anti-and proapoptotic proteins, such as caspases, PUMA, Bax, and Bak. However, this hypothesis still needs to be further analyzed. One limitation of this study is that the assays were executed in only one breast cancer cell line. It would be important to evaluate the effect of F. crocata extracts on a larger number of tumor and non-tumor cell lines. Furthermore, the effect of pure metabolites isolated from F. crocata extracts, such as lupeol, Stigmastan, and others, has not, to our knowledge, been tested. This effect will be evaluated in a subsequent study. Our observations comprise, to our knowledge, a first approach to investigating the possible use of extracts of F. crocata as alternative or complementary therapy against cancer. Nonetheless, their cytotoxicity and molecular mechanisms must be analyzed in more study models.

Conclusions
The leaf extract of F. crocata decreases the proliferation capacity of MDA-MB-231 triple-negative breast cancer cells, decreasing the S and G2/M cell cycle phases, and inducing apoptosis possibly through an p53-independent pathway. By fractionating the extract, the antiproliferative activity against MDA-MB-231 cells can be further potentiated. These findings provide information on the biological activity of F. crocata extracts and suggest their potential use as an alternative or complementary therapy against triple-negative breast cancer. Nevertheless, more studies are required to determine the components responsible for this biological activity, as well as the molecular and cellular mechanisms involved.